Abstract

BACKGROUND:

Injuries to the spinal cord often result in severe functional deficits that, in case of incomplete injuries, can be partially compensated by axonal remodeling. The corticospinal tract (CST), for example, responds to a thoracic transection with the formation of an intraspinal detour circuit. The key step for the formation of the detour circuit is the sprouting of new CST collaterals in the cervical spinal cord that contact local interneurons. How individual collaterals are formed and refined over time is incompletely understood.

METHODOLOGY/PRINCIPAL FINDINGS:

We traced the hindlimb corticospinal tract at different timepoints after lesion to show that cervical collateral formation is initiated in the first 10 days. These collaterals can then persist for at least 24 weeks. Interestingly, both major and minor CST components contribute to the formation of persistent CST collaterals. We then developed an approach to label single CST collaterals based on viral gene transfer of the Cre recombinase to a small number of cortical projection neurons in Thy1-STP-YFP or Thy1-Brainbow mice. Reconstruction and analysis of single collaterals for up to 12 weeks after lesion revealed that CST remodeling evolves in 3 phases. Collateral growth is initiated in the first 10 days after lesion. Between 10 days and 3-4 weeks after lesion elongated and highly branched collaterals form in the gray matter, the complexity of which depends on the CST component they originate from. Finally, between 3-4 weeks and 12 weeks after lesion the size of CST collaterals remains largely unchanged, while the pattern of their contacts onto interneurons matures.

CONCLUSIONS/SIGNIFICANCE:

This study provides a comprehensive anatomical analysis of CST reorganization after injury and reveals that CST remodeling occurs in distinct phases. Our results and techniques should facilitate future efforts to unravel the mechanisms that govern CST remodeling and to promote functional recovery after spinal cord injury.

Population analysis of hindlimb CST collateral formation at different timepoints after SCI.

(A–C) Reconstruction of hindlimb CST collaterals (black) from 5 consecutive sections in the cervical spinal cord of control mice (A) and of mice perfused 10 days (B) and 24 weeks (C) following SCI. (D) Quantification of the total numbers of collaterals emerging from all CST components in the cervical gray matter of control mice and of mice at different timepoints following SCI. (E–G) Confocal images of main CST (BDA, yellow) and the adjacent gray matter (Neurotrace, blue; border shown by dashed white line) in control mice (E) and in mice perfused 10 days (F, arrow indicates CST collateral emerging from main CST) and 24 weeks (G, arrow indicates CST collateral emerging from main CST) following SCI. (H) Quantification of the number of collaterals emerging from the main CST component at different timepoints following SCI. (I–K) Confocal images of the minor dorso-lateral CST (BDA, yellow) and the adjacent gray matter (Neurotrace, blue; border shown by dashed white line) in control mice (I) and in mice perfused 10 days (J) and 24 weeks (K, arrow indicates CST collateral emerging from dorso-lateral CST) following SCI. (L) Quantification of the number of collaterals emerging from the minor dorso-lateral CST component at different timepoints following SCI. (M–O) Confocal images of the minor ventral CST (BDA, yellow) and the adjacent gray matter (Neurotrace, blue; border shown by dashed white line) in control mice (M, arrow indicates ventral CST fiber) and in mice perfused 10 days (N) and 24 weeks (O, arrow indicates collateral emerging from ventral CST) following SCI. (P) Quantification of the number of collaterals emerging from the minor ventral CST component at different timepoints following SCI. Asterisks indicate significance compared to the unlesioned controls. Pound signs indicate significance compared to the 10-day timepoint. Scale bar in A (also for B,C), 500 µm; in M (also for E–O), 100 µm.

Reconstruction of individual hindlimb CST collaterals at different timepoints after spinal cord injury.

(A–C) Reconstruction of individual collaterals (blue asterisks indicate the entry point of the collateral in the gray matter) emerging from the main dorsal (A) and the minor ventral (B) and dorso-lateral (C) CST components at 10 days following SCI. (D–F) Reconstruction of individual collaterals (green) emerging from the main dorsal (D) and the minor ventral (E) and dorso-lateral (F) CST components at 4 weeks following SCI. (G–I) Reconstruction of individual collaterals (red) emerging from the main dorsal (G) and the minor ventral (H) and dorso-lateral (I) CST components at 12 weeks following SCI. (J–L) Quantification of the total collateral length (J), the number of branchpoints/collateral (K) and the number of boutons/collateral (L) measured in individually reconstructed collaterals at different timepoints after SCI. Blue bars, 10-day timepoint; green bars, 3-week timepoint; red bars, 12-week timepoint. Asterisks indicate significant differences compared to the 10-day timepoint. Pound signs indicate significant differences between collaterals emerging from different CST components at 3 weeks (green) and 12 weeks (red) after injury. Scale bar in A (also for B–I), 50 µm.

(A–B) Confocal images of bassoon immmunostaining (green) in the cervical spinal cord of mice with a traced hindlimb CST (BDA, red) perfused 10 days (A) and 3 weeks following SCI (B). Yellow arrows indicate boutons that were immunoreactive for bassoon, white arrows indicate those that were not. (A′–A′″) Single plane confocal image of the boutons boxed in A showing the collateral (A′; BDA, white), bassoon immunostaining (A″; white) and the overlay (A′″; BDA, red; bassoon, green) at 10 days after SCI. (B′–B′″) Single plane confocal image of the bouton boxed in B showing the collateral (B′; BDA, white), bassoon immunostaining (B″, white) and the overlay (B′″; BDA, red; bassoon, green) at 3 weeks after SCI. (C) Quantification of the number of boutons on hindlimb CST collaterals that were immunopositive for bassoon at 10 days and 3 weeks following SCI in the cervical cord. The percentages were normalized to the expression pattern in the lumbar cord (L) of control animals (which was set to 100%). (D–E) Confocal images of synapsin I immunostaining in the cervical spinal cord of mice with a traced hindlimb CST (BDA, red) perfused 10 days following SCI (D) and at 3 weeks post-injury (E). Yellow arrows indicate boutons that were immunoreactive for synapsin I, white arrows indicate those that were not. (D′–D′″) Single plane confocal image of the bouton boxed in D showing the collateral (D′, BDA, white), the synapsin I staining (D″; white) and the overlay (D′″; BDA, red; synapsin I, green) at 10 days after SCI. (E′–E′″) Single plane confocal image of the bouton boxed in E showing the collateral (E′; BDA, white), the bassoon staining (E″; white) and the overlay (D′″; BDA, red; synapsin I, green) at 3 weeks after SCI. (F) Quantification of the number of boutons on hindlimb CST collaterals that were immunnopositive for synapsin I at 10 days and 3 weeks following SCI in the cervical cord. The percentages were normalized to the expression pattern in the lumbar cord (L) of control animals (which was set to 100%). (G) Quantification of the co-expression of bassoon and synapsin I in boutons of CST collaterals of animals perfused at 3 weeks after injury (expressed as percentages of all immunoreactive boutons). Scale bar in A (also for B, D, E), 10 µm and in A′ (also for A″–E′″), 3 µm.

(A,B) Confocal images of contacts (arrows, defined as boutons in apposition to neuronal cell bodies) between hindlimb CST collaterals (YFP, green) and the cell bodies of cervical interneurons (Neurotrace, red) at 4 weeks (A) and 12 weeks (B) following SCI. (C) Quantification of the number of contacts a given hindlimb CST collateral makes with the cell body of a single interneuron at different timepoints after SCI as well as in the lumbar spinal cord of unlesioned animals. (D) Quantification of the percentage of Neurotrace (NT)-stained interneurons contacted by collaterals emerging from the different CST components at multiple timepoints following the lesion. Asterisks indicate significant difference compared to main CST collaterals. Scale bar in A (also for B), 15 µm.

Scheme illustrating the formation of cervical collaterals derived from the main CST (upper row) and the minor dorsolateral (2nd row) and ventral (3rd row) CST components at 10 days (blue), 3–4 weeks (green) and 12 weeks (red) after SCI. Bottom row illustrates the refinement over time of the contacts between CST collaterals and cervical interneurons.

(A) Confocal image of a cross-section of the thoracic (T8) spinal cord of a mouse perfused 12 weeks after dorsal hemisection (counterstained with Neurotrace). Dashed line indicates lesion border. (B) Schematic representation of the location of the different CST components (highlighted in different shades of green) in relation to this lesion (outlined by dashed line from A). Scale bar in A, 200 µm.